1. Field of the Invention
The present invention relates to a measurement method and a measurement apparatus utilizing attenuated total reflection. In the measurement method and apparatus, a light beam is totally reflected at an interface between a thin film layer, which is in contact with a sample, and a dielectric block to generate an evanescent wave. Then, a variation in the intensity of the totally reflected light beam is measured to analyze a sample.
2. Description of the Related Art
Free electrons in metal vibrate in a group, and a compression wave is generated. The compression wave is called a plasma wave. When the compression wave, which is generated on a surface of the metal, is quantized, it is called a surface plasmon.
Conventionally, various surface plasmon measurement apparatuses utilizing a phenomenon of the surface plasmon, excited by a light wave, have been proposed to analyze the properties of substances to be measured. Particularly, an apparatus using a system called the Kretschmann configuration is well-known among the surface plasmon measurement apparatuses (Japanese Unexamined Patent Publication No. 6(1994)-167443).
Basically, the surface plasmon measurement apparatus using the above-mentioned system includes a dielectric block, for example, in a prism shape, a metal film, which is formed on a surface of the dielectric block and brought into contact with a substance to be measured, such as sample liquid, a light source for generating a light beam, an optical system for causing the light beam to enter the dielectric block at various angles so that total reflection conditions are satisfied at the interface between the dielectric block and the metal film, and a light detection means for detecting a surface plasmon resonance state, namely an attenuated total reflection state, by measuring the intensity of the light beam, which is totally reflected at the interface.
A relatively thin light beam may be caused to enter the interface by changing the incident angle so that the light beam enters the interface at various incident angles as described above. Alternatively, a relatively thick light beam in a convergence light state or divergence light state may be caused to enter the interface so that the light beam includes components that enter the interface at various angles. In the former case, a reflection angle of the light beam changes according to the change in the incident angle of the light beam, which enters the interface, and the light beam may be detected by a small light detector, which moves synchronously with the change in the reflection angle. Alternatively, the light beam may be detected by an area sensor, which extends along the direction of the change in the reflection angle. In the latter case, the light beams may be detected by an area sensor, which extends in a direction so that each of the light beams reflected at various reflection angles may be detected.
In the surface plasmon measurement apparatus structured as described above, when the light beam is caused to enter the metal film at a specific incident angle, which is larger than or equal to a total reflection angle, an evanescent wave is generated. The electric field of the evanescent wave is distributed in the substance to be measured, which is in contact with the metal film. Then, surface plasmon is excited by the evanescent wave at the interface between the metal film and the substance to be measured. When a wave number vector of the evanescent wave is equal to the wave number of the surface plasmon, and the wave numbers are matched, the evanescent wave and the surface plasmon resonate. Then, light energy is transferred to the surface plasmon. Therefore, the intensity of the light, which was totally reflected at the interface between the dielectric block and the metal film, sharply decreases. Generally, the decrease in the intensity of the light is detected as a dark line by the light detection means. The resonance as described above occurs only if the incident beam is p polarized. Therefore, it is necessary to set in advance so that the light beam enters the interface in p polarization.
If the wave number of the surface plasmon is obtained based on an incident angle, at which attenuated total reflection (ATR) occurs, namely an attenuated total reflection angle θsp, the dielectric constant of the substance to be measured may be obtained. Specifically, if the wave number of the surface plasmon is Ksp, an angular frequency of the surface plasmon is ω, the speed of light in a vacuum is c, and the dielectric constants of the metal and the substance to be measured are εm and εs, respectively, the following relationship is satisfied:
            k      sp        ⁡          (      ω      )        =            ω      c        ⁢                                                      ɛ              m                        ⁡                          (              ω              )                                ⁢                      ɛ            s                                                              ɛ              m                        ⁡                          (              ω              )                                +                      ɛ            s                              
Specifically, if the attenuated total reflection angle θsp is obtained, the dielectric constant εs, namely, properties related to the refractive index of the substance to be measured, may be obtained. The attenuated total reflection angle θsp is an incident angle, at which the intensity of the reflection light decreases.
In the surface plasmon measurement apparatus as described above, an array-shaped light detection means as disclosed in U.S. Pat. No. 6,577,396 may be used to accurately measure the attenuated total reflection angle θsp in a wide dynamic range. In the light detection means, a plurality of light receiving elements is arranged in a predetermined direction. The plurality of light receiving elements is arranged in a direction so that each component of the light beam, which is totally reflected at various reflection angles at the interface, is received by a different light receiving element.
When the light receiving elements are arranged as described above, a differential means for differentiating a light detection signal, output from each of the light receiving elements of the array-shaped light detection means, with respect to the arrangement direction of the light receiving elements, is provided in many cases. Properties related to the refractive index of the substance to be measured is obtained based on a differential value, output by the differential means.
Further, a leaky mode measurement apparatus, described in “Spectrum Researches”, Journal of The Spectroscopical Society of Japan, volume 47, No. 1, 1998, pp 21-23 and 26-27, is also known as a similar measurement apparatuses utilizing the attenuated total reflection (ATR). Basically, the leaky mode measurement apparatus includes a dielectric block, for example, in a prism shape, a clad layer formed on a face of the dielectric block, and an optical waveguide layer, which is formed on the clad layer and brought into contact with sample liquid. The leaky mode measurement apparatus also includes a light source for generating a light beam, an optical system for causing the light beam to enter the dielectric block at various angles so that total reflection conditions are satisfied at the interface between the dielectric block and the clad layer, and a light detection means for detecting an excitation state of a guided wave mode by measuring the intensity of the light beam, totally reflected at the interface. The excitation state is an attenuated total reflection state.
In the leaky mode measurement apparatus structured as described above, when the light beam is caused to enter the clad layer at an incident angle, larger than or equal to a total reflection angle, through the dielectric block, the light beam is transmitted through the clad layer. After the light beam is transmitted through the clad layer, only light with a specific wave number, which has entered at a specific incident angle, propagates in a guided wave mode in the optical waveguide layer. When the guided wave mode is excited as described above, a substantial part of the incident light is absorbed by the optical waveguide layer. Accordingly, the attenuated total reflection occurs, in which the intensity of light, totally reflected at the interface, sharply decreases. The wave number of the guided wave light depends on the refractive index of the substance to be measured on the optical waveguide layer. Therefore, if the specific incident angle, at which the attenuated total reflection occurs, is found, the refractive index of the substance to be measured and the properties of the substance to be measured, related to the refractive index, may be analyzed.
In the leaky mode measurement apparatus, the array-shaped light detection means, as described above, may be also used to detect the position of a dark line in a reflection light, generated by the attenuated total reflection. Further, the differential means as described above is also applied to the leaky mode measurement apparatus in many cases.
Further, the surface plasmon measurement apparatus and the leaky mode measurement apparatus, as described above, are used in random screening to find a specific substance, which will be combined with a desired sensing substance, in a research field such as drug discovery. In this case, a sensing substance, as the substance to be measured, is fixed on the thin film layer (the metal film in the case of the surface plasmon measurement apparatus, and the clad layer and the optical waveguide layer in the case of the leaky mode measurement apparatus). Sample liquid is added onto the sensing substance. In the sample liquid, various kinds of objects to be examined are dissolved in a solvent. Then, the attenuated total reflection angle θsp is measured at predetermined time intervals.
If the object to be examined in the sample liquid is an object that binds with the sensing substance, as time passes, the object to be examined binds with the sensing substance, and the refractive index of the sensing substance changes. Therefore, if the attenuated total reflection angle θsp is measured at predetermined time intervals to measure whether the attenuated total reflection angle θsp has changed, the binding state of the object to be measured and the sensing substance is measured. It is possible to judge whether the object to be measured is the specific substance, which binds with the sensing substance. As examples of the combination of the specific substance and the sensing substance, as described above, there are a combination of an antigen and an antibody and a combination of an antibody and an antibody. Specifically, a rabbit anti-human IgG (immunoglobulin G) antibody, as the sensing substance, may be fixed to the surface of the thin film layer, and a human IgG antibody may be used as the specific substance.
When measuring the binding state of the object to be measured and the sensing substance, it is not always necessary to detect the attenuated total reflection angle θsp itself. For example, the sample liquid is added to the sensing substance, and the variation in angle of the attenuated total reflection angle θsp after the addition may be measured. The binding state may be measured based on the magnitude of the variation in angle. If the array-shaped light detection means and the differential means, as described above, are applied to a measurement apparatus utilizing attenuated total reflection, the variation of a differential value reflects the variation in angle of the attenuated total reflection angle θsp. Therefore, the binding state of the sensing substance and the object to be measured may be measured based on the variation of the differential value.
In the measurement method and apparatus utilizing the attenuated total reflection as described above, sample liquid including a solvent and an object to be examined is supplied to a measurement chip by dropping. The measurement chip is cup-shaped or petri-dish-shaped, and a sensing substance is fixed on a thin film layer, formed on the bottom of the measurement chip in advance. Accordingly, the variation in angle of the attenuated total reflection angle θsp is measured.
When the sample liquid is supplied to the measurement chip, and the sensing substance and the object to be measured are bound with each other, the refractive index of the sensing substance changes. Accordingly, the attenuated total reflection angle θsp changes. Therefore, if after predetermined time has passed from the beginning of the measurement, the variation in angle of the attenuated total reflection angle θsp from the beginning of the measurement is obtained, it is possible to judge whether the object to be examined binds with the sensing substance. Further, if the object to be examined is bound with the sensing substance, it is possible to analyze the binding state of the object to be examined and the sensing substance, or the like. However, strictly speaking, the variation in angle of the attenuated total reflection angle θsp, detected by the measurement apparatus, does not accurately reflect the variation of the refractive index, caused by the combination of the sensing substance and the object to be examined. It is known that there is a difference in the measurement sensitivity depending on the thickness of the metal film in the measurement chip, the surface shape (coarseness) of the measurement chip, or the like.
Therefore, a sensitivity calibration method has been proposed to correct the difference in the sensitivity of each measurement chip. According to the method, every time that the measuring chip is changed, only solvent (buffer) is supplied to the measuring chip before measuring the properties of the sample liquid. The sensitivity of the measuring chip is detected based on a signal, which is output by measuring the properties of the solvent, and the sensitivity is calibrated.
Further, U.S. Patent Application Publication No. 20030075697 proposes a method for calibrating an actual measurement value based on a calibration curve. In this method, the properties of a plurality of kinds of reference samples of known refractive indices are measured, and the calibration curve is obtained based on the measurement result. U.S. Patent Application Publication No. 20030075697 discloses the use of liquid of a known refractive index as the reference sample and the use of a measurement chip (calibration tool), in which a solid material of a known refractive index (dielectric constant) is fixed (evaporated) on a thin film layer of the measurement chip.
However, the inventor of the present invention has found out that signal variation behaviors are different between a biomolecule, which is an object to be measured, and the buffer. Therefore, even if the sensitivity is calibrated by using the buffer, there is a difference in signals when the properties of an actual biomolecule are measured.
FIG. 7 is a graph illustrating a variation in the signal values of attenuated total reflection with respect to a physical property value k (an imaginary number, which is an absorption term, in a refractive index). Here, the signal is a SPR (surface plasmon resonance) signal, and its shift angle is the signal value. The physical property value k is proportional to the conductivity σ of metal. In FIG. 7, the signal values of the buffer are represented by circles (◯), and the signal values of protein, which is a biomolecule, are represented by crosses (X). The signal values are standardized with respect to a reference physical property value, which is set to 1. As illustrated in FIG. 7, even if the physical property value k of metal changes by ±10%, there is substantially no difference in the signal values of the buffer. In contrast, there is a difference of approximately ±20% in the signal values of the protein, which is the biomolecule. Therefore, it is obvious that the sensitivity calibration method, using the buffer, according to the related art is not sufficient to detect the biomolecule.
Further, even if liquid having a known refractive index is used as a reference sample, as disclosed in U.S. Patent Application Publication No. 20030075697, when the properties of an actual biomolecule are measured, there seems to be a difference in signals in a similar manner to the case of correcting the sensitivity by using the buffer. Therefore, there is a problem that when a calibration tool is used, the sensitivity is not calibrated for each of the measurement chips.